Lymphocyte numbers are regulated both by responses to conventional exogenous antigens and by stimulation by endogenous peptide/MHC complexes. This joint regulation permits individuals to maintain a broad repertoire of specificities, allowing responses against a vast array of foreign substances and, at the same time, providing a pattern of memory based on the immunization history of the individual. The study of the process of lymphocyte dynamics that underlies this regulation requires a multidisciplinary approach, aimed both at the molecular underpinnings of the processes through which lymphocytes survive and proliferate and a systemics/ computational biology approach to appreciate the overall mechanisms governing total numbers of lymphocytes of distinct phenotype and distinct specificity. Emphasis has been placed on four aspects of this problem: the priming of naive CD4 T cells, the dynamics of lymphocyte memory, the mechanisms underlying CD4 T cell depletion in HIV infection, and the process of homeostatic proliferation. During the past year, it has been shown that primary responses are highly dependent upon the number of precursor cells that can respond to antigenic challenge. Using both real time PCR and flow cytometric analysis to measure the response when TCR transgenic cells are transferred to intact recipients, it has been shown that responses of CD4 T cells are not linear above 300 in the total population of lymph node CD4 T cells. When limiting numbers of cells are transferred, the expansion factor of immunization is ~1000. When larger numbers of precursors are present, the factor of expansion may be as low as 20. The limitation in expansion does not result from a smaller fraction of cells responding but rather, at least in part, from early death of expanded cells when large numbers are present. Thus, the traditional approach of transferring millions of naive TCR transgenic cells to obtain information about primary immune responses cannot be expected to yield results that reflect physiologic responses. Our analysis of responses made by small numbers of transferred cells provides an approach to assess physiologic responses of naive cells. Death is not due to Fas-mediated killing but may reflect competition for key cytokines or other growth/ survival factors or more likely for access to pMHC complexes on dendritic cells. The degree of expasion of naive and memory CD4 T cells is very much determined by the presence of adjuvants or molecules that can engage the innate immune system. Thus, the use of pure protein antigens causes little or no expasion even when small numbers of precursor cells are present whereas factors of expasion up to or exceeding 1000-fold are obtained with the addition of LPS. Recently, it has been shown that the most potent inducer of expansion of naive and of memory CD4 T cells is the cytokine IL-1. This cytokine displays its powerful effect even when wild-type T cells are transferred into IL-1 receptor knock out recipients, implying that the T cell is the direct target of IL-1 in this system. The effect of IL-1 is not mimicked by IL-18, even though IL-18 receptors are expressed on many memory cells and many of the signlaing pathways through which the IL-1 and IL-18 receptors function are similar. Similarly, IL-1 is far more potent that LPS, suggesting that TLR-signaling pathways are less potent for this purpose that IL-1 signaling pathways. The high degree of potency of IL-1 in determiing the factor of expasnion of naive and memory CD4 T cells suggests that adjuvant development should focus on the IL-1 pathway. Analysis of the ?steady state? proliferation of memory CD4 T cells reveals that it is similar in conventional and germfree mice. Nonetheless, analysis of BrdU labeling and delabeling kinetics and proliferative potential of memory cells upon transfer to intact syngeneic recipients implies that much of this proliferation reflects "burst-like" proliferative events rather than stochastic ?turnover". Thus, analysis of syngeneic mice that received transfers of CFSE-labeled CD44bright/ CD62Ldull (memory) cells one week after the transfer reveals that more than 10% of the surviving cells have completed multiple divisions (often more than 7), implying that at steady state a comparable fraction of memory CD4 T cells have just completed a burst of cell divisions. The similarlity of the prolifertative rate in conventional and germfree mice suggests that this is not driven by intestinal microflora or conventional antigens and that it may represent self-reactivity. Utilizing multiparameter flow cytometric analysis, we have identified populations of cells with distinctive markers that appear to correspond to central and peripheral memory cells and that now allow careful tracing of the movement of cells through various cellular compartments in the course of responses to cognate antigenic stimulation. Most strikingly, there is a population of CD4 T cells that express high levels of CD44 and CD45RB, as well as CD62L. These cells are turning over relatively rapidly, consistent with the cells representing a population of memory cells. There expression of CD62L implies that they are central memory cells; thus will make possible direct analysis of central memory cells using a single phenotypic marker (very high levels of CD45RB) and should make it possible to follow immunization events in vivo in a more decisive manner. Utilizing the techniques of recombineering, we are preparing indicator mice for expression of three key ?homeostasis-determining? cytokines, IL-7, TSLP and IL-15. These indicator mice should allow the detailed analysis of the cellular sites of production of these cytokines and the precise regulation of expressions of these cytokines. Working with colleagues at the Oregon Regional Primate Center, we are analyzing lymphocyte dynamics in SIV-infected macaques and have obtained evidence for CD4 T cell populations that decay at very distinct rates in the period after their burst-like expansion. Laboratory of Immunology scientists and our colleagues have shown the critical role that aberrant immune activation in lentiviral infection plays in the decay of CD4 T cell number and in the degradation of immunocompetence in SIV-infected macaques and HIV-infected humans. The ongoing activation of the immune system caused direclty or indirectly by infection appears to be of particualr importance in the loss of potential effector cells during the chronic phase of the infection. A collaboration has been initiated with professor G. Bacherov of the Russian Academy of Sciences to use computational methods to analyze lymphocyte dynamics. With the growing numbers of tools to subdivdie lymphoycte populations and to asses their function, such computational analysis will be of great value in the effort to develop realistic models of the dynamics of these cells.